In vitro test system to evaluate xenobiotics as immune-modulators of drug transport and metabolism in human hepatocytes

ABSTRACT

A kit and method for evaluating in vitro the effect of a xenobiotic (e.g., a biologic drug) on drug metabolism in hepatocytes. The kit comprises a first culture of hepatocytes, a portion of in vitro xenobiotic-stimulated biological sample, and instructions for incubating the first culture of hepatocytes with the portion of in vitro xenobiotic-stimulated biological sample, and analyzing the activity, expression, or a combination thereof of a biomarker in the hepatocytes to evaluate the effect of the xenobiotic on drug metabolism in the hepatocytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 13/481,295, filed May 25, 2012, which claims the priority benefit ofU.S. Provisional Patent Application Ser. No. 61/490,931, filed May 27,2011, entitled IN VITRO TEST SYSTEM TO EVALUATE XENOBIOTICS ASIMMUNE-MODULATORS OF DRUG TRANSPORT AND METABOLISM IN HUMAN HEPATOCYTES,each of which is incorporated by reference in its entirety herein.

BACKGROUND

1. Field of the Invention

The present disclosure relates to in vitro methods of evaluatingxenobiotics and their effect on drug transport and metabolism.

2. Description of Related Art

There is an increasing number of biological drugs (“biologics”) beingdeveloped as alternative therapeutics to traditional small moleculedrugs. There are significant differences between these macromoleculebiologics and small molecule drugs, including the ways in which theyreact with the body. Cytochrome P450 (CYP) enzymes are the major enzymesinvolved in small molecule drug metabolism and bioactivation. Severalbiologics are known to suppress CYP activity and result in smallmolecule drug toxicity following their administration. For example,influenza and flu vaccination has been shown to suppress CYP1A2 andCYP2C9 activity and thereby impair theophylline and warfarin clearance,respectively, resulting in theophylline and warfarin toxicity. The fluvaccine is also known to impair aminopyrine metabolism afterinoculation. Some bacterial endotoxins (e.g., LPS) are known to affectthe oral drug clearance of antipyrine, hexobarbital, and theophylline inotherwise healthy individuals. For example, following LPS treatment,changes in antipyrine (AP) clearance were found in humans and correlatedwith changes in TNF and IL-6.

SUMMARY

Since biologics affect drug metabolizing enzymes, which can change thesystemic exposure to normally hepatically-cleared drugs leading to aloss of efficacy, drug toxicity, and/or exaggerated pharmacology, thereis a need in the art for improved methods of evaluating potentialdrug-drug interactions, particularly in the pre-clinical setting andparticularly between biologics and small molecule drugs. There is afurther need for in vitro test systems that better reflect actual invivo metabolism and potential interactions. An in vitro test system isdescribed herein which allows assessment of xenobiotics asimmune-mediated or direct modulators of liver function. Broadly, thetest system includes a culture of a biological sample and a separateculture of hepatocytes. In the biological sample culture, the immunesystem cells respond to exposure to a xenobiotic by secreting signalingmolecules such as cytokines, interleukins, interferons, and growthfactors. The xenobiotic-stimulated fraction is separated and thentransferred to the culture of hepatocytes. The immune systemcell-mediated effects of xenobiotics on various biomarkers, such as drugtransporters and drug-metabolizing enzymes are then measured in thehepatocytes. The method is particularly suited for determining andanalyzing potential for (adverse) drug interactions, especially ofbiologics, when co-administered with traditional small molecule drugs.

A method of evaluating the effect of a xenobiotic on drug metabolism inhepatocytes is provided. The method comprises providing a first cultureof hepatocytes, and providing a portion of a xenobiotic-stimulatedbiological sample. The portion of the xenobiotic-stimulated biologicalsample is transferred to the first culture of hepatocytes, and theactivity and/or expression levels of a biomarker are analyzed in thehepatocytes. The term “biomarker” is used herein to refer to substances(such as peptides, molecules, proteins, etc.) that have predictive valuefor the effect of a given xenobiotic in a biological system. The termincludes drug transporters, drug-metabolizing enzymes, or a combinationthereof, and the like.

In related embodiments, a further method of evaluating xenobiotics asimmune-modulators of drug transport and metabolism in hepatocytes isprovided. The method comprises providing a first culture of a biologicalsample and a first culture of hepatocytes. The first biological sampleculture is exposed to a xenobiotic for a period of time to yield axenobiotic-stimulated biological sample. A portion of thexenobiotic-stimulated biological sample is transferred to the firstculture of hepatocytes. The immune system-mediated effects of thexenobiotic on drug transporters and drug-metabolizing enzymes in thehepatocytes are evaluated by analyzing the activity and/or expressionlevels of a biomarker in the hepatocytes.

A kit for evaluating the effect of a xenobiotic on drug metabolism inhepatocytes is also provided. The kit comprises hepatocytes, a portionof a xenobiotic-stimulated biological sample, and instructions forincubating the hepatocytes with the portion of xenobiotic-stimulatedbiological sample and analyzing the activity and/or expression levels ofa biomarker in the hepatocytes. In one or more embodiments the portionof a xenobiotic-stimulated biological sample is plasma comprisingcytokines, which has been separated from xenobiotic-stimulated wholeblood. In related embodiments, the portion of a xenobiotic-stimulatedbiological sample is a fraction comprising cytokines, which has beenseparated from xenobiotic-stimulated bone marrow.

A further method of evaluating the effect of a xenobiotic on drugmetabolism in hepatocytes is also provided. The method comprisesproviding a first culture of hepatocytes and a xenobiotic-stimulatedbiological sample. The xenobiotic-stimulated biological sample istransferred to the first culture of hepatocytes, and the activity,expression, or combination thereof of a biomarker in said hepatocytes isanalyzed. In some embodiments, the biological sample is isolatedperipheral mononuclear blood cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart of processes for the in vitro evaluation ofxenobiotics as immune modulators of drug transport and metabolism inhepatocytes;

FIG. 2 shows fluorescent microscopy images of (A) Kupffer cells stainedwith anti-CD68 antibody; and (B) hepatocytes and Kupffer cells stainedwith a nuclear stain;

FIG. 3 shows graphs of the effects of plasma obtained from LPS- orcontrol-stimulated whole blood on CYP1A2 in human hepatocytes (n=3) fromExample 1;

FIG. 4 shows graphs of the effects of plasma obtained from LPS- orcontrol-stimulated whole blood on CYP2B6 in human hepatocytes (n=3) fromExample 1;

FIG. 5 shows graphs of the effects of plasma obtained from LPS- orcontrol-stimulated whole blood on CYP3A4 in human hepatocytes (n=3) fromExample 1;

FIG. 6 shows graphs of the effects of LPS on CYP activity in humanhepatocytes co-cultured with Kupffer cells (n=3) from Example 1;

FIG. 7 shows graphs of the effects of anti-CD28 mAb and LPS on cytokinerelease in whole blood from Example 2;

FIG. 8 shows graphs of the effects of plasma obtained from anti-CD28antibody or control-stimulated whole blood on CYP1A2 in humanhepatocytes (n=3) from Example 2;

FIG. 9 shows graphs of the effects of plasma obtained from anti-CD28antibody or control-stimulated whole blood on CYP2B6 in humanhepatocytes (n=3) from Example 2;

FIG. 10 shows graphs of the effects of plasma obtained from anti-CD28antibody or control-stimulated whole blood on CYP3A4 in humanhepatocytes (n=3) from Example 2; and

FIG. 11 shows graphs of the effects of plasma obtained from anti-CD28antibody or control-stimulated whole blood on drug transporters in humanhepatocytes (n=3) from Example 2.

DETAILED DESCRIPTION

Embodiments disclosed herein are concerned with methods of evaluatingxenobiotics as immune-modulators of drug transport and metabolism inhepatocytes. The term xenobiotics is used herein to refer to a compoundthat is foreign to a living organism, such as a drug. The termencompasses biologics as well as small molecule drugs, although thedisclosed embodiments are particularly suited to biologic-typexenobiotics and certain small molecules that have pro-inflammatorycytokine effects. The term “biologics” is used herein to refer totherapeutic compounds (e.g., biologic medicines and drugs) that arecreated primarily through biological processes, rather than via purechemical synthesis (like traditional small molecule drugs). Biologicscan be composed of sugars, proteins, nucleic acids, or combinationsthereof, and also include living cells and tissues. Biologics can beisolated from a variety of natural sources, such as human, animal, ormicroorganism. They can also be produced using recombinant DNAtechniques. Exemplary biologics that can be evaluated using the presentembodiments, include hormones (e.g., insulin), growth factors (e.g.,erythropoeitin, GM-CSF, GH), cytokines (e.g., interferons,interleukins), receptors (e.g., soluble TNFα receptor), antibodies(e.g., anti-cytokine receptors, MAbs conjugated to small molecule drugsor poisons), enzymes (e.g., streptokinase), oligonucleotides (antisense)(e.g., fomivirsen for CMV retinitis), aptomers, iRNA or syntheticpeptides, vaccines (e.g., influenza), biological extracts (e.g., beevenom), and the like.

The methods comprise providing a biological sample, which has beencollected from a subject. In one or more embodiments, the subject is amammal, including non-human mammals (e.g., rodents, dogs) and humans. Inrelated embodiments, the subject is a human. In one or more embodiments,the sample can be from either a healthy (normal) or diseased subject.Exemplary biological samples include tissues which yield immune-mediatedeffects upon exposure to a xenobiotic, such as whole blood, bone marrow,and even isolated peripheral blood mononuclear cells (PBMCs). In one ormore embodiment, the biological sample is whole blood. In use, a wholeblood culture is prepared from at least a portion of the biologicalsample. In one or more embodiments, the culture comprises the collectedwhole blood and an anticoagulant (e.g., heparin, EDTA, sodium citrate)in a suitable container. In some embodiments, the culture does notinclude an anticoagulant (i.e., the whole blood is not mixed with ananticoagulant, and may, in some embodiments, be allowed to clot).Regardless, the whole blood culture is then incubated with a selectedxenobiotic. In more detail, the xenobiotic is typically dispersed in avehicle or carrier to produce a xenobiotic solution. Suitable vehiclesor carriers will depend upon the xenobiotic, but can include saline,phosphate buffer, and the like. The concentration of the xenobiotic inthe solution should be selected to be clinically relevant, which meansthat it closely approximate actual in vivo plasma concentrations, andwill typically range from about 5% to about 2,000%, and more preferablyfrom about 10% to about 1,000% of Cmax (the maximal concentration inplasma in vivo). The xenobiotic solution is then added to the wholeblood culture. In one or more embodiments, the whole blood culture andxenobiotic are incubated for at least about 12 hours, and preferablyfrom about 12 to about 48 hours, at a temperature of approximately 37°C., to yield stimulated whole blood. The immune system cells in theblood, such as PBMCs, are stimulated by the xenobiotic. In response, thecells produce signaling molecules such as growth factors, growthhormones chemokines, interferons, interleukins, (referred tocollectively herein as “cytokines”), which are secreted or released intothe plasma. In one or more embodiments, the methods described herein canbe characterized as yielding artificially-stimulated blood (as opposedto natural in vivo stimulation) and related artificially-stimulatedfractions.

The plasma is then separated from the xenobiotic-stimulated whole bloodto yield xenobiotic-stimulated plasma. Any suitable separation techniquecan be used. In one or more embodiments, the xenobiotic-stimulated wholeblood is centrifuged at about 600 x g for about 10 minutes. Thesupernatant (i.e., plasma) is then aspirated from the pellet. Thexenobiotic-stimulated plasma can be analyzed directly or transferred toa second container and stored under suitable conditions until needed foranalysis. In one or more embodiments, at least a portion of thexenobiotic-stimulated plasma is analyzed for the effects of thexenobiotic on the expression of cytokines. Typically, cytokines assayedin plasma from xenobiotic-stimulated whole blood cultures include, butare not limited to, interleukins IL-1B, IL-2, IL-6, IL-8, IL-10, andIL-12p70, granulocyte-macrophage colony-stimulating factor (GM-CSF),interferons (IFN-γ), tumor necrosis factor (TNF-α), and the like. In oneor more embodiments, the plasma can be analyzed using an ELISA-basedassay. Fluorescent or luminescent (e.g., chemiluminescent) probes can beused to detect the assay results. A portion of the whole blood can alsobe analyzed for the expression of cytokine genes by isolating cytokinemRNAs.

In the method, an aliquot (portion) of the xenobiotic-stimulated plasmais added to a culture of hepatocytes in a suitable cell culture medium(e.g., MCM+ (modified Chee's medium)). In one or more embodiments, thehepatocyte culture is actually a co-culture ofhepatocytes and Kupffercells (liver macrophages). The hepatocytes and/or Kupffer cells can befresh or thawed (e.g., from cryopreserved cells), and isolated fromhuman and/or animal livers. Pooled hepatocyte cultures prepared frommultiple sources may also be used. A cell “source,” as used herein,refers cells obtained from various donors, biopsies, tissue resectionsfrom different tissue samples or different tissue sources, differentanimals harboring cells (species), or primary, secondary, immortalized,or transformed cells. The cells may be derived from any mammaliansource, including human, porcine, simian, canine, feline, bovine,equine, ovine, leporine, or murine sources, among others. In one or moreembodiments, the hepatocytes are primary human hepatocytes. In relatedembodiments, the Kupffer cells are isolated from human livers. Cells maybe obtained from a single source at two or more different times,combined, and cryopreserved. Such cells would still be considered to beprepared from a “single source.” Cells from different sources includethose obtained from mammalian cells of different genders, genotypes,ages, races (e.g., Caucasian, etc.), enzymatic or metabolic activities,species, or disease or health states (e.g., hepatocytes of hepatitisvirus-infected liver, hepatocytes of HIV-1 infected liver, hepatocytesof healthy liver, hepatocytes of cigarette smokers, hepatocytes ofindividuals suffering from cirrhosis of the liver, or from otherdiseases or conditions, such as rheumatoid arthritis, cancer, and/orCrohn's disease, etc.). Cells from different sources are particularlydesired for producing pooled preparations.

The terms “pooled” preparation or “pooling,” as used herein, refer to acomposition of cells that results from the combination of cells frommore than one source, and generally comprises such cells suspended in aculture medium. The cells of such pooled preparations may be randomlyselected, or may be specifically selected to provide the pooledpreparation with a desired level of one or more metabolic activities(such as for example, a preparation of hepatocytes having a desiredlevel of enzymatic activity, as described herein), or a desired cellcharacteristic (such as, for example, a preparation of hepatocytesderived from sources of a particular gender, genotype, age, race, orhealth state). For example, pooled hepatocyte preparations may beformulated so as to provide a preparation having the metabolicactivities of an “average” hepatocyte sample or a preparation whosehepatocyte enzyme functions approximate the hepatocyte enzyme functionsof freshly isolated hepatocytes. Such metabolic activities may include,for example, some or all of the following enzymatic activities:bupropion hydroxylase, amodiaquine N-dealkylase, diclofenac 4′-hydroxylase, coumarin 7-hydroxylase (COUM), dextromethorphanO-demethylase (DEX), 7-ethoxycoumarin O-deethylase (ECOD), mephenytoin4-hydroxylase (MEPH), testosterone 6(β)-hydroxylase (TEST), tolbutamide4-hydroxylase (TOLB), phenacetin O-deethylase (PHEN), chlorzoxazone6-hydroxylase (CZX), or activities responsible for the phase IImetabolism of 7-hydroxycoumarin (7-HCG (glucuronidase) and 7-HCS(sulfatase).

Those skilled in the art will recognize that hepatocyte-like cells mayalso be used in the disclosed embodiments. Hepatocyte-like cells includethose derived from stem cells or an immortal cell line, that otherwisehave enzymatic activity (see above) characteristic of hepatocytes aswell as the ability to respond to cytokines (e.g., have appropriatefunctional receptors on their cells). Thus, the term hepatocyte is usedherein to encompass such hepatocyte-like cells.

In some embodiments, the xenobiotic-stimulated plasma and hepatocyteculture are incubated for at least about 24 hours, and preferably fromabout 48 to about 72 hours at about 37° C. In one or more embodiments,the culture media and plasma is removed every 24 hours and replaced withfresh culture media and fresh stimulated plasma. It will be appreciatedthat exposure of the Kupffer cells to plasma containing cytokines mayalso result in their activation, causing the production and release ofadditional signaling molecules.

The cells in the hepatocyte culture are then analyzed to evaluate theimmune system-mediated effects of the xenobiotic on biomarkers in thehepatocytes. More specifically, in one or more embodiments the cells inthe hepatocyte culture are further incubated with a selected enzymemarker substrate. Exemplary marker substrates are listed in the tablebelow.

TABLE I Enzyme Marker substrate (reaction) CYP1A2 Phenacetin(O-dealkylation) CYP2A6 Coumarin (7-hydroxylation) CYP2B6 Bupropion(hydroxylation) CYP2C8 Amodiaquine (N-dealkylation) CYP2C9 Diclofenac(4′-hydroxylation) CYP2C19 S-Mephenytoin (4′-hydroxylation) CYP2D6Dextromethorphan (O-demethylation) CYP2E1 Chlorzoxazone(6-hydroxylation) CYP3A4/5 Testosterone (6β-hydroxylation) CYP3A4/5Midazolam (1′-hydroxylation) UGT 7-Hydroxycoumarin (glucuronidation)SULT 7-Hydroxycoumarin (sulfonation)In some embodiments, the hepatocyte culture is incubated in situ withthe marker substrate for at least about 5 minutes (preferably from about10 to 30 minutes) at about 37° C. The disappearance of substrate and/orappearance of metabolites can be analyzed using any suitable technique,including liquid chromatography with mass spectrometry (LC/MS),including tandem mass spectrometry (LC/MS/MS). Enzymatic activity canalso be analyzed using microsomes isolated from the hepatocytes.

In one or more embodiments, the hepatocytes can be isolated from theculture to determine enzyme mRNA levels and activities or expressionlevels of drug metabolizing enzymes and drug transporters or otherbiomarkers after exposure to the xenobiotic-stimulated plasma. Inrelated embodiments, total RNA can be isolated from the hepatocytesusing known methods, such as TRIzol (Invitrogen) or TRI (Sigma-Aldrich),as well as column-based methods like RNAeasy (Qiagen) to determine theeffect of the xenobiotic on the enzyme mRNA levels. Suitable RNAanalysis techniques are known in the art, and include PCR (e.g., RT-PCR)techniques. In one or more embodiments, enzymatic activity assays (e.g.,ELISA) can also be carried out to determine the activities of drugmetabolizing enzymes and drug transporters. The test systems describedherein are particularly suited for predicting the in vivo effect axenobiotic may have on a particular enzymatic pathway known to beassociated with a given small molecule drug. In this way, potential invivo drug interactions can be predicted in the pre-clinical setting.Functional analysis can also be carried out on the hepatocytes todetermine additional effects on the hepatocyte drug transporters andenzymes.

In one or more embodiments, the cells from the xenobiotic-stimulatedplasma and hepatocyte culture can be incubated with a small moleculedrug of interest. Depending upon the metabolic stability of the smallmolecule drug, the cells can be incubated with the drug for a timeperiod of at least about 30 minutes, and typically from about 1 to about4 hours. The metabolism of the small molecule drug by the hepatocytes(which may or may not have impaired enzymatic activity) can then beevaluated. This test system is particularly suited for studyingpotential biologic interactions with a small molecule having an unknownmetabolism (i.e., where the specific enzymes and drug transportersinvolved in its metabolism are unknown or not well established). Again,the in vitro system provides a predictive model of in vivo metabolismand interaction.

It will be appreciated that the present methods can be applied to othertissues producing cytokines in vivo, such as bone marrow, as well asisolated PBMCs. The process will be the same for the whole blooddescribed above, except that instead of separating plasma afterincubation of the sample with the xenobiotic, the supernatant (which isthe fraction containing the cytokines) will be collected. Thisxenobiotic-stimulated fraction will then be applied to the hepatocyteculture.

In some embodiments, the methods also further comprise appropriatepositive and negative controls. In one or more embodiments, a positivecontrol for cytokine release can be prepared using a knowncytokine-eliciting compound to ensure that the biological sample iscapable of producing an adequate or robust cytokine response and/or thatthis function has not been impaired. The method is almost identical tothat described above for the xenobiotic, except that a compound withknown cytokine-stimulating effects is used in lieu of the xenobiotic.Briefly, the biological sample is incubated with the selected compound.A portion of the sample (e.g., the plasma or other cytokine-containingfraction) is then separated, as described above, and incubated with aseparate culture of hepatocytes (and/or Kupffer cells) following thesame protocol used for the xenobiotic-stimulated whole blood and plasma.The resulting cells are also similarly analyzed. Exemplary compounds foruse as the positive control include LPS, CpG DNA, PHA (phytohemagglutinin), Candida albicans soluble extract, zymosan, and the like. Insome embodiments, where the xenobiotic is a conjugated protein, it maybe desirable to also test each protein separately as a positive controlor a negative control.

In one or more embodiments, a negative control for cytokine release canbe prepared following substantially the same method described above.However, instead of incubating the biological sample with a xenobiotic,the sample is incubated with the selected vehicle or carrier (withoutany xenobiotic). A portion of the sample (e.g., the plasma) is thenseparated, as described above, and incubated with a separate culture ofhepatocytes (and Kupffer cells) following the same protocol used for thexenobiotic-stimulated whole blood and plasma. The resulting cells arealso similarly analyzed.

In one or more embodiments, a direct negative control can be prepared byincubating the culture of hepatocytes (and Kupffer cells) directly withthe vehicle or carrier (without any blood, plasma, or xenobiotic). Thisprocess is followed by analysis of the cells using the same methodsdescribed above.

In further embodiments, a direct xenobiotic sample can be prepared byincubating the culture of hepatocytes (and Kupffer cells) directly withthe xenobiotic (without any blood or plasma), followed by analysis ofthe cells using the same methods described above.

In yet further embodiments, an additional positive control can beprepared by incubating the culture of hepatocytes (and Kupffer cells)directly with a known hepatic enzyme suppressor, such as IL-6 (withoutany blood or plasma), followed by analysis of the cells using the samemethods described above.

One of the benefits of the disclosed embodiments is the ability toevaluate multiple cytokines that are released in response to thexenobiotic in a single assay. This multiplexing allows not just theevaluation of multiple cytokines at once, but also assessment ofpotential differences in cytokine behavior in different cytokinecombinations. More specifically, in prior art methods, cytokines thatare released upon stimulation from a xenobiotic are not subsequentlyapplied to hepatocytes. Rather, these cytokines are identified, and thensynthesized or purchased from commercially-available sources. They arethen examined, one at a time or in limited combinations, in drugmetabolism studies. These processes do not evaluate or provide anyinformation regarding the interaction between multiple cytokines andtheir effect on drug metabolism, as would be encountered in the in vivosystem. Thus, current the CYP suppression by cytokines in current invitro methods does not accurately reflect CYP suppression by cytokinesin vivo. Thus, the present in vitro test systems provide a morerealistic view of the in vivo effects of xenobiotics and druginteraction. The disclosed embodiments allow for an in vitro modeling ofthe interactions among hepatocytes and immune system cells in theperipheral blood and the liver upon xenobiotic exposure. The methodsprovide several benefits towards assessment of the effects ofxenobiotics. These include the evaluation of the drug-drug interactionpotential between small molecule drugs and biologic drugs that may be aconsequence of a direct interaction by the xenobiotic with hepatocytesand Kupffer cells in vivo. Furthermore, xenobiotics that affecthepatocytes indirectly through the immune system cells in blood can alsobe evaluated. The disclosed methods assess the potential of xenobiotics,such as drug candidates, to cause drug-drug interaction by alteringexpression of drug-metabolizing enzymes and drug transporters. Thesedata can streamline the process of pre-clinical drug development.

Additional advantages of the various embodiments of the invention willbe apparent to those skilled in the art upon review of the disclosureherein and the working examples below. It will be appreciated that thevarious embodiments described herein are not necessarily mutuallyexclusive unless otherwise indicated herein. For example, a featuredescribed or depicted in one embodiment may also be included in otherembodiments, but is not necessarily included. Thus, the presentinvention encompasses a variety of combinations and/or integrations ofthe specific embodiments described herein.

As used herein, the phrase “and/or,” when used in a list of two or moreitems, means that any one of the listed items can be employed by itselfor any combination of two or more of the listed items can be employed.For example, if a composition is described as containing or excludingcomponents A, B, and/or C, the composition can contain or exclude Aalone; B alone; C alone; A and B in combination; A and C in combination;B and C in combination; or A, B, and C in combination.

The present description also uses numerical ranges to quantify certainparameters relating to various embodiments of the invention. It shouldbe understood that when numerical ranges are provided, such ranges areto be construed as providing literal support for claim limitations thatonly recite the lower value of the range as well as claim limitationsthat only recite the upper value of the range. For example, a disclosednumerical range of about 10 to about 100 provides literal support for aclaim reciting “greater than about 10” (with no upper bounds) and aclaim reciting “less than about 100” (with no lower bounds).

EXAMPLES

The following examples set forth methods in accordance with theinvention. It is to be understood, however, that these examples areprovided by way of illustration and nothing therein should be taken as alimitation upon the overall scope of the invention.

Example 1 Analysis of Xenobiotic Effect on CYP Enzymes in Hepatocytes

Whole blood was collected from one healthy volunteer into vacutainerscontaining 15 USP sodium heparin/mL of blood. Blood cultures werestimulated ex vivo with lipopolysaccharides (LPS) 50 ng/mL in normalsaline for 24 hrs at 37° C. Following the stimulation period, plasma wasseparated from the blood cells by centrifugation and stored at −80° C.Levels of cytokines IL-1B, IL-2, IL-6, IL-8, IL-10, IL-12p70, GM-CSF,INF-γ, TNF-α in the plasma from the LPS-stimulated whole blood wereanalyzed with the Sector Imager 24-00 and Pro-inflammatory 9-Plex kitmanufactured by Meso Scale Discovery.

Co-cultures of primary human hepatocytes and Kupffer cells were preparedand stained with anti-CD68 mAb or a nuclear stain(4′,6-diamidino-2-phenylindole, DAPI). Images were taken usingfluorescence microscopy are shown in FIG. 2. The ratio of macrophage tohepatocyte was approximately 1:50.

Three concentrations (10, 20, or 50%) of LPS-stimulated plasma in a cellculture medium were prepared. Three concentrations (10, 20, or 50%) ofplasma from saline-stimulated whole blood were prepared in a cellculture medium. Three concentrations (10, 25, or 50 ng/mL) of LPS in acell culture medium were also prepared.

To assess effects of LPS on the expression of CYP enzymes in humanhepatocytes, three co-cultures of human hepatocytes and Kupffer cellsfrom three separate livers were treated daily for 72 hours with: (1)saline (negative control); (2) one each of the three concentrations ofplasma from LPS-stimulated whole blood (positive control for cytokinerelease); (3) one each of the three concentrations of saline-stimulatedplasma (negative control for cytokine release); (4) one each of threeconcentrations of LPS (direct xenobiotic test); or (5) interlukin-6 (10ng/mL), a known suppressor of human CYP3A4 (additional positivecontrol). After treatment, the cells in each group were incubated insitu with the appropriate marker substrates for the analysis ofphenacetin O-dealkylation (marker for CYP1A2), bupropion hydroxylation(marker for CYP2B6) and testosterone 6β-hydroxylation (marker forCYP3A4/5) by LC/MS/MS. Following the in situ incubation, the samehepatocytes from the same treatment groups were harvested with TRIzol toisolate RNA, which was analyzed by qRT-PCR to assess the effect of theplasma from LPS-stimulated whole blood on CYP1A2, CYP2B6 and CYP3A4 mRNAlevels.

Treatment of co-cultures of hepatocyte and Kupffer cells with IL-6caused anticipated decreases in CYP1A2, CYP2B6, and CYP3A4 activity(FIGS. 3-5 and 8-10). Treatment of co-cultures of hepatocyte and Kupffercells with plasma from LPS-stimulated WB caused a decrease in CYP 1A2,CYP2B6, and CYP3A4 activity (FIGS. 3-5). The effects of IL-6, LPS, andplasma from LPS-stimulated whole blood on enzymatic activities inhepatocytes were accompanied by expected corresponding changes in thelevels of specific mRNAs. Taken together these results indicated thatenzyme suppression rather than inhibition was the mechanism responsiblefor the observed changes in the activity of CYP enzymes (FIGS. 3-5).Treatment of co-cultures of hepatocyte and Kupffer cells with LPS causeddecrease in CYP1A2 and CYP3A4, but not CYP2B6 activity (FIG. 6).

Under the conditions of this study where the IL-6 control causedanticipated decreases in CYP activities, treatment with up to 50% ofplasma derived from LPS-stimulated WB resulted in comparable (CYP2B6 andCYP3A4) or greater (CYP1A2) decrease in the activity of CYP enzymes thanthe IL-6 control. These results indicate that if LPS is administered toa human patient, cytokines released from whole blood will affect drugmetabolizing cells in the liver, meaning that the individual will have areduced capacity for metabolizing other co-administered drugs (and apossibility for drug interaction).

The results demonstrate that the test system allows in vitro assessmentof xenobiotics as immune-modulators of liver function.

Example 2 Monoclonal Antibody Xenobiotic

In this Example, the same procedures used in Example 1 were followed,except that anti-CD28 antibody was used as the xenobiotic. LPS was alsoused as an additional positive control. The changes in pro-inflammatorycytokines observed upon treatment of whole blood with LPS or anti-CD28antibody are illustrated in FIG. 7. Treatment of co-cultures ofhepatocyte and Kupffer cells with plasma from anti-CD28antibody-stimulated WB caused a decrease in CYP 1 A2, CYP2B6, and CYP3A4activity (FIGS. 8-10). Treatment of co-cultures of hepatocyte andKupffer cells with anti-CD28 antibody had little or no effect on CYP andCYP3A4, and CYP2B6 activity (FIGS. 8-10). The effects of IL-6,anti-CD28antibody, and plasma from anti-CD28 antibody whole blood on enzymaticactivities in hepatocytes were accompanied by expected correspondingchanges in the levels of specific mRNAs. Taken together these resultsindicated that enzyme suppression rather than inhibition was themechanism responsible for the observed changes in the activity of CYPenzymes (FIGS. 8-10).

Under the conditions of this study where the IL-6 control causedanticipated decreases in CYP activities, treatment with up to 50% ofplasma derived from anti-CD28 antibody-stimulated whole blood resultedin comparable decrease in the activity of CYP enzymes as the IL-6control. These results indicate that if anti-CD28 antibody isadministered to a human patient, cytokines released from whole bloodwill affect drug metabolizing cells in the liver, meaning that theindividual will have a reduced capacity for metabolizing otherco-administered drugs (and a possibility for drug interaction).

The effects of treating hepatocytes with plasma from the LPS- oranti-CD28 antibody-treated whole blood resulted in decrease in the mRNAlevels of hepatic transporters OATP1B1, OATP2B1 and NTCP (FIG. 11).

We claim:
 1. A kit for evaluating in vitro the effect of a xenobiotic ondrug metabolism in hepatocytes, said kit comprising: a first culture ofhepatocytes; an in vitro xenobiotic-stimulated biological sample; andinstructions for incubating said first culture of hepatocytes with saidin vitro xenobiotic-stimulated biological sample and analyzing theactivity, expression, or a combination thereof of a biomarker in saidhepatocytes to evaluate the effect of said xenobiotic on drug metabolismin said hepatocytes.
 2. The kit of claim 1, wherein said hepatocytes arecryopreserved hepatocytes.
 3. The kit if claim 1, wherein said firstculture of hepatocytes is a co-culture of hepatocytes and Kupffer cells.4. The kit if claim 1, wherein said hepatocytes are human hepatocytes.5. The kit if claim 1, wherein said hepatocytes are pooled hepatocytes.6. The kit of claim 1, wherein said in vitro xenobiotic-stimulatedbiological sample is a portion of in vitro xenobiotic-stimulatedbiological sample prepared by exposing a first culture of saidbiological sample to said xenobiotic in vitro to yield axenobiotic-stimulated biological sample, and separating said portion ofsaid xenobiotic-stimulated biological sample.
 7. The kit of claim 6,wherein said portion of in vitro xenobiotic-stimulated biological sampleis plasma that has been separated from in vitro xenobiotic-stimulatedwhole blood.
 8. The kit of claim 6, wherein said portion of in vitroxenobiotic-stimulated biological sample is a cytokine-containingfraction that has been separated from in vitro xenobiotic-stimulatedbone marrow.
 9. The kit of claim 6, wherein said portion of in vitroxenobiotic-stimulated biological sample is a cytokine-containingfraction that has been separated from in vitro xenobiotic-stimulatedperipheral blood mononuclear cells.
 10. The kit of claim 1, wherein saidxenobiotic is a biologic drug.
 11. The kit of claim 10, wherein saidbiologic drug is selected from the group consisting of hormones, growthfactors, cytokines, receptors, antibodies, enzymes, oligonucleotides,aptomers, synthetic peptides, and vaccines.
 12. The kit of claim 1,further comprising: a second culture of hepatocytes; a sample of saidxenobiotic; and instructions for incubating said second culture ofhepatocytes directly with said sample of said xenobiotic, and analyzingthe activity, expression, or combination thereof of a biomarker in saidsecond culture of hepatocytes.
 13. The kit of claim 12, wherein saidsample of said xenobiotic comprises said xenobiotic dispersed in acarrier, said kit further comprising a negative control, said negativecontrol comprising: a third culture of hepatocytes; a sample of saidcarrier; and instructions for incubating said third culture ofhepatocytes directly with said sample of said carrier, analyzing theactivity, expression, or combination thereof of a biomarker in saidthird culture of hepatocytes.
 14. The kit of claim 13, said kit furthercomprising a fourth culture of hepatocytes; an in vitrocarrier-stimulated biological sample; and instructions for incubating aportion of said in vitro carrier-stimulated biological sample with saidfourth culture of hepatocytes.
 15. An in vitro method of evaluating theeffect of a xenobiotic on drug metabolism in hepatocytes, said methodcomprising: providing a first culture of hepatocytes; providing an invitro xenobiotic-stimulated biological sample, wherein said biologicalsample is isolated peripheral blood mononuclear cells; transferring saidxenobiotic-stimulated biological sample to said first cultureofhepatocytes; and analyzing the activity, expression, or combinationthereof of a biomarker in said hepatocytes.
 16. The method of claim 15,wherein said providing a xenobiotic-stimulated biological samplecomprises: providing a first culture of isolated peripheral bloodmononuclear cells; and exposing said first culture of isolatedperipheral blood mononuclear cells to a xenobiotic for a period of timeto yield said xenobiotic-stimulated biological sample.
 17. The method ofclaim 15, wherein said xenobiotic is a biologic drug.
 18. The method ofclaim 17, wherein said biologic drug is selected from the groupconsisting of hormones, growth factors, cytokines, receptors,antibodies, enzymes, oligonucleotides, aptomers, synthetic peptides, andvaccines.
 19. The method of claim 15, wherein said hepatocytes are humanhepatocytes.
 20. The method of claim 15, wherein said hepatocytes arepooled hepatocytes.